Media concentrate technology

ABSTRACT

The present invention relates to a method of subgrouping media formulations into stable, compatible components that can then be solubilized at high concentrations (10× to 100×). Concentrated solutions of the ingredient containing subgroups can be used to prepare media formulations. Concentrated culture media formulations (2-10×) or 1× cell culture media can be prepared according to the present invention by mixing a sufficient amount of the concentrated subgroup solutions with a sufficient amount of a diluent (water, buffer, etc.). 
     The present invention further relates to stabilization of glutamine by complexing or mixing glutamine with divalent metal cations in solution.

This application is a continuation of application Ser. No. 08/124,508,filed Sep. 21 1993, U.S. Pat. No. 5,474,931 which is a continuation ofapplication Ser. No. 07/716,264, filed Jun. 17, 1981 abandoned.

FIELD OF THE INVENTION

The present invention relates to the field of cell culture media andspecifically to media ingredients subgrouped selectively intoconcentrated, compatible formulations. Such concentrated formulationsare stable and can be used to prepare cell culture media.

BACKGROUND OF THE INVENTION

Cell culture medium provides the nutrients necessary to maintain andgrow cells in a controlled, artificial and in vitro environment.Characteristics and compositions of the cell culture media varydepending on the particular cellular requirements. Important parametersinclude osmolarity, pH, and nutrient formulations.

Media formulations have been used to grow a number of cell typesincluding animal, plant and bacterial cells. Cells grown in culturemedia catabolize available nutrients and produce useful biologicalsubstances such as monoclonal antibodies, hormones, growth factors andthe like. Such products have therapeutic applications and, with theadvent of recombinant DNA technology, cells can be engineered to producelarge quantities of these products. Thus, the ability to grow cells invitro is not only important for the study of cell physiology, it isnecessary for the production of useful substances which may nototherwise be obtained by cost-effective means.

Cell culture media formulations have been well documented in theliterature and a number of media are commercially available. Typicalnutrients in cell culture media include amino acids, salts, vitamins,trace metals, sugars, lipids and nucleic acids. Often, particularly incomplex media compositions, stability problems result in toxic productsand/or lower effective concentrations of required nutrients, therebylimiting the functional life-span of the culture media. For instance,glutamine is a constituent of almost all media that are used inculturing of mammalian cells in vitro. Glutamine decomposesspontaneously into pyrrolidone carboxylic acid and ammonia. The rate ofdegradation can be influenced by pH and ionic conditions but in cellculture media, formation of these breakdown products cannot be avoided(Tritsch et al., Exp. Cell Research 28:360-364(1962)).

Wang et al. (In Vitro 14:(8):715-722 (1978)) have shown thatphotoproducts such as hydrogen peroxide, which are lethal to cells, areproduced in Dulbecco's Modified Eagle's Medium (DMEM). Riboflavin andtryptophan or tyrosine are components necessary for formation ofhydrogen peroxide during light exposure. Since most mammalian culturemedia contain riboflavin, tyrosine and tryptophan, toxic photoproductsare likely produced in most cell culture media.

To avoid these problems, researchers make media on an "as needed" basis,and avoid long term storage of the culture media. Commercially availablemedia, typically in dry power form, serves as a convenient alternativeto making the media from scratch, i.e., adding each nutrientindividually, and also avoids some of the stability problems associatedwith liquid media. However, only a limited number of commercial culturemedia are available, except for those custom formulations supplied bythe manufacturer.

Although dry powder media formulations may increase the shelf-life ofsome media, there are a number of problems associated with dry powderedmedia, especially in large scale application. Production of large mediavolumes requires storage facilities for the dry powder media, not tomention the specialized media kitchens necessary to mix and weigh thenutrient components. Due to the corrosive nature of dry powder media,mixing tanks must be periodically replaced.

There exists a need to lower the cost of production of biologicalsubstances. Efficient and cost effective methods to stabilize liquidcell culture media as well as the development of convenient methods toproduce 1× media formulations would be an important development in thefield of cell culture media technology.

SUMMARY OF THE INVENTION

The present invention relates to a method of subgrouping mediaformulations into stable, compatible components that can then besolubilized at high concentrations (10×to 100×) in solution containing,for example, water, acid, alkali or alcohol, depending on the subgroup.Any media formulation can be divided into one or more of the sixsubgroups of the present invention. Specific subgroups include an acidsoluble subgroup, a glutamine containing subgroup, an alkali solublesubgroup, an alcohol soluble subgroup, a weak acid-base soluble subgroupand a supplement-containing subgroup.

According to the present invention, one of ordinary skill in the art maydivide ingredients of a cell culture media into one of the subgroups ofthe present invention based on the physical and chemical properties(physicochemical properties) of each ingredient. Ingredients of culturemedia typically include amino acids, salts, vitamins, trace metals,sugars, lipids, nucleic acids and the like. These ingredients havedifferent solubility and stability characteristics. Based on thesecharacteristics, a classification scheme has been developed to permitthe compatible subgrouping of media ingredients.

The invention thus relates to a method of subgrouping compatible mediaingredients. Media ingredients, subgrouped in this manner, are stableand remain soluble even at high concentrations. Such concentratedsolutions of the ingredient containing subgroups can then be used toprepare diluted media formulations suitable for growing or maintainingcells. Concentrated culture media formulations (2-10×) or 1× cellculture media can be prepared according to the present invention byadmixing sufficient amount of the concentrated subgroup solutions with asufficient amount of a diluent (water, buffer, etc.). Thus, the presentinvention is directed to a method of preparing a cell culture mediacomprising:

(a) dividing the compatible ingredients of a cell culture media into oneor more subgroups, such subgroups comprising:

(i) an acid soluble subgroup,

(ii) a weak acid-base soluble subgroup,

(iii) a glutamine-containing subgroup,

(iv) an alcohol soluble subgroup,

(v) an alkali-soluble subgroup, and

(vi) a supplement-containing subgroup;

(b) dissolving each of the subgroups of compatible ingredients in acarrier to give concentrated solutions of the subgroup in the respectivecarrier; and

(c) admixing a sufficient amount of each of the subgroups obtained instep (b) with a sufficient amount of a diluent to produce the cellculture media.

Furthermore, the present invention is specifically directed to a methodof preparing RPMI-1640 media comprising:

(a) dividing the ingredients of RPMI-1640 media into

(i) an acid soluble subgroup,

(ii) a weak acid-base soluble subgroup, and

(iii) a glutamine-containing subgroup;

(b) dissolving each of the subgroups of compatible ingredients in acarrier to give concentrated solutions of the subgroup in the respectivecarrier; and

(c) admixing a sufficient amount of each of the subgroups obtained instep (b) with a sufficient amount of a diluent to produce the RPMI-1640media.

This method can also be used to make DMEM media. That is, theingredients of DMEM media can be divided into an acid soluble subgroup,a weak acid-base soluble subgroup and a glutamine-containing subgroupwhich are combined at some later time and diluted to give a 1×formulation.

The present invention further relates to stabilization of glutamine bycomplexing or mixing glutamine with divalent metal cations in solution.Thus, the present invention is also directed to a composition of mattercomprising glutamine and a divalent cation in an amount effective tostabilize the glutamine.

Unexpectedly, the present invention provides for the preparation ofmedia at dramatically reduced cost. The cost reductions are due to thefollowing factors: The media concentrates of the present invention maybe produced with much smaller production facilities since the large stirtanks required for 1× medias are not required. In addition, the mediaconcentrates of the present invention may be prepared on an as neededbasis using "just in time" production techniques which reduce inventory,storage and labor costs. The time required for the preparation andshipping of the media may be reduced from 6-8 weeks to as little as oneday. In addition, the media concentrates of the present invention may beprepared without the use of ball milling machines since each of thesubgrouped ingredients is readily soluble. In addition, the mediasubgroups of the present invention may be stored at room temperaturewhich eliminates the need for refrigeration. In addition, the presentinvention allows for the preparation of concentrates which may be usedto prepare very large quantities of 1× media (100,000 liters or greater)which require only one quality control test compared to multiple qualitycontrol tests for multiple batches produced according to the prior art.Importantly, the media concentrates of the present invention are muchmore consistent between batches since the subgrouped components are morestable. Thus, the present invention is a great advance in the art ofmedia formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows L-glutamine stabilization in solution. Various ratios ofL-glutamine to divalent cations were tested and the stability ofL-glutamine was determined.

FIG. 2 shows the monitored pH and osmolarity over time of RPMI-1640media prepared by the continuous admixture of a sufficient amount ofconcentrated subgroups with a sufficient amount of water to make a 1×media formulation.

FIG. 3 shows the monitored level of L-glutamine and glucose over time.The samples were taken from a mixing chamber during the continuousadmixture of concentrated subgroups with water.

FIG. 4 shows a kit comprising large containers containing concentratedsubgroups. The concentrated subgroups may be admixed and diluted to givea total of 10,000 liters of media.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the description that follows, a number of terms conventionally usedin the field of cell culture media are utilized extensively. In order toprovide a clear and consistent understanding of the specification andclaims, and the scope to be given such terms, the following definitionsare provided.

Ingredients. The term "ingredients" refer to any compound, whether ofchemical or biological origin, that can be used in cell culture media tomaintain or promote the growth or proliferation of cells. The terms"component", "nutrient" and "ingredient" can be used interchangeably andare all meant to refer to such compounds. Typical ingredients that areused in cell culture media include amino acids, salts, metals, sugars,lipids, nucleic acids, hormones, vitamins, fatty acids, proteins and thelike. Other ingredients that promote or maintain growth of cells invitro can be selected by those of skill in the art, in accordance withthe particular need.

Cell Culture. By "cell culture" is meant cells or tissues that aremaintained, cultured or grown in an artificial, in vitro environment.

Culture Vessel. Glass, plastic or metal containers of various sizes thatcan provide an aseptic environment for growing cells are termed "culturevessels."

Cell Culture Media. The phrases "cell culture media" or "culture media"or "media formulation" refer to a nutritive solution for culturing orgrowing cells. The ingredients that compose such media may varydepending on the type of cell to be cultured. In addition to nutrientcomposition, osmolarity and pH are considered important parameters ofculture media.

Compatible Ingredients. Each ingredient used in cell culture media hasunique physical and chemical characteristics. By "compatibleingredients" is meant those media nutrients which can be maintained insolution and form a "stable" combination. A solution containing"compatible ingredients" is said to be "stable" when the ingredients donot degrade or decompose substantially into toxic compounds, or do notdegrade or decompose substantially into compounds that can not beutilized or catabolized by the cell culture. Ingredients are alsoconsidered "stable" if degradation can not be detected or whendegradation occurs at a slower rate when compared to decomposition ofthe same ingredient in a 1× cell culture media formulation. Glutamine,for example, in 1× media formulations, is known to degrade intopyrrolidone carboxylic acid and ammonia. Glutamine in combination withdivalent cations are considered "compatible ingredients" since little orno decomposition can be detected over time.

Compatibility of media ingredients, in addition to stabilitymeasurements, are also determined by the "solubility" of the ingredientsin solution. The term "solubility" or "soluble" refers to the ability ofan ingredient to form a solution with other ingredients. Ingredients arethus compatible if they can be maintained in solution without forming ameasurable or detectable precipitate. Thus, the term "compatibleingredients" as used herein refers to the combination of particularculture media ingredients which, when mixed in solution either asconcentrated or 1× formulations, are "stable" and "soluble."

1× Formulation. A cell culture media is composed of a number ofingredients and these ingredients vary from media to media. A "1×formulation" is meant to refer to any aqueous solution that containssome or all ingredients found in a cell culture media. The "1×formulation" can refer to, for example, the cell culture media or to anysubgroup of ingredients for that media. The concentration of aningredient in a 1× solution is about the same as the concentration ofthat ingredient found in the cell culture formulation used formaintaining or growing cells. Cell culture media used to grow cells is a1× formulation by definition. When a number of ingredients are present(as in a subgroup of compatible ingredients), each ingredient in a 1×formulation has a concentration about equal to the concentration ofthose ingredients in a cell culture media. For example, RPMI 1640culture media contains, among other ingredients, 0.2 g/l L-arginine,0.05 g/l L-asparagine, and 0.02 g/l L-aspartic acid. A "1× formulation"of these amino acids, which are compatible ingredients according to thepresent invention, contains about the same concentrations of theseingredients in solution. Thus, when referring to a "1× formulation," itis intended that each ingredient in solution has the same or about thesame concentration as that found in the cell culture media beingdescribed. The concentrations of media ingredients in a 1× formulationare well known to those of ordinary skill in the art, See Methods ForPreparation of Media, Supplements and Substrate For Serum-Free AnimalCell Culture Alan R. Liss, New York (1984), which is incorporated byreference herein in its entirety. The osmolarity and/or pH, however, maydiffer in a 1× formulation compared to the culture media, particularlywhen fewer ingredients are contained by the 1× formulation.

10× Formulation. A "10× formulation" refers to a solution wherein eachingredient in that solution is about 10 times more concentrated than thesame ingredient in the cell culture media. RPMI 1640 media, for example,contains, among other things, 0.3 g/l L-glutamine. By definition, a "10×formulation" contains about 3.0 g/l glutamine. A "10× formulation" maycontain a number of additional ingredients at a concentration about 10times that found in the 1× culture media. As will be apparent, "25×formulation," "50× formulation" and "100× formulation" designatesolutions that contain ingredients at about 25, 50 or 100 foldconcentrations, respectively, as compared to a 1× cell culture media.Again, the osmolarity and pH of the media formulation and concentratedformulation may vary.

A. Media Concentrate Technology

The Media Concentrate Technology of the present invention is based onthe subgrouping of media ingredients into stable, compatible componentsthat can then be solubilized into concentrated formulations (10×-100×)by the dissolution in an appropriate quantity of a carrier such aswater, aqueous acid, alcohol or aqueous alkali, depending on theparticular subgroup. Such solutions are referred to herein as a"carrier". Under appropriate conditions, these subgroups can bereconstituted (admixed) to produce a concentrated (greater than 1× )cell culture media formulation, such as a 2× to 10×, or reconstituteddirectly to a 1× cell culture media formulation. The concentrate mediatechnology of the present invention is related to the subgrouping ofcompatible components, production of liquid concentrates of suchsubgroups, and mixing sufficient amounts of these concentrated subgroupswith a sufficient amount of a diluent to produce a desired mediaformulation.

Cell culture media formulations are made up of amino acids, salts,vitamins, trace metals, sugars, lipids, nucleic acids, etc. Each ofthese components has different solubility and stability properties.Amino acids, for example, (except for glutamine) are soluble and verystable under acidic conditions (pH of about 1.0) while vitamins (exceptfor thiamine and ascorbic acid) are solubilized under dilute acid oralkaline conditions (pH of about >4.0). Based on the physical propertiesof media ingredients, a classification scheme was developed that permitsthe subgrouping of any media formulation which can be used to preparestable liquid concentrates.

The present invention defines six subgroups in which compatibleingredients from any cell culture media can be divided. The ingredientsof a given cell culture media formulation can be separated into at leastone of the subgroups defined by the present invention. Each subgroup,containing the compatible media ingredients, can then be dissolved in aliquid carrier or maintained in dry form. The type of liquid carrier andthe method used to dissolve the grouped ingredients into solution varydepending on the subgroup and can be determined by one of ordinary skillin the art with no more than routine experimentation.

Preferably, the solutions comprising the subgrouped ingredients ere moreconcentrated than the concentration of the same ingredients in a 1×media formulation. The grouped ingredients are preferably 25 fold moreconcentrated (25× formulation) and most preferably 50 fold moreconcentrated (50× formulation). Higher concentrated formulations can bemade provided that the ingredients remain soluble and stable.

Once the media ingredients have been divided into the one or more of thesix subgroups and prepared as separate concentrated solutions, anappropriate (sufficient) amount of each concentrate is combined with adiluent to produce a 1× media formulation. Typically, the diluent usedis water but other solutions including aqueous buffers, aqueous salinesolution, or other aqueous solutions containing particular mediaingredients may be used according to the invention.

The culture media of the present invention is typically sterilized toprevent unwanted contamination of the cell culture media. Sterilizationmay be accomplished, for example, by pasteurization after admixing theconcentrated ingredients to produce a sterile culture media.Alternatively, each concentrated subgroup may be sterilized and storedas a sterile solution. These sterile concentrates can then be mixed witha sterile diluent to produce a concentrated 1× sterile mediaformulation.

One of a number of commonly used sterilization techniques may be used tosterilize the concentrated subgroups or the media formulations of thepresent invention. Sterilization techniques will vary depending on theproperties of the solutions to be sterilized. Typical sterilizationtechniques, according to the present invention, include heat and/orfilter sterilization. Other types of sterilization will be readilyapparent to one of ordinary skill in the art.

B. Type of Media

Any type of cell culture media can be made according to the presentinvention. Culture media formulations are well-known in the literatureand a number are commercially available. New media formulations andknown media formulations which have been modified can also be preparedaccording to the present invention. That is, the media ingredients canbe divided into compatible subgroups as defined in the presentinvention. The subgroups of these ingredients can then be concentratedin solution and, if desired, sterilized.

Examples of cell culture media that can be prepared according to thepresent invention include, but are not limited to, DMEM, RPMI-1640,DMEM/F12, IMDM, MEM, M199, McCoy's 5A, and F12 Nutrient Mixture(GIBCO/BRL CATALOGUE & PREFERENCE GUIDE (1990)).

The present invention thus provides a means for making one or moreconcentrated solutions of compatible ingredients, which when sufficientamounts are mixed in combination with sufficient amounts of diluent, acell culture media may be produced.

Any ingredient employed in a cell culture media can be divided into oneor more of the six subgroups of the present invention. Although the listof ingredients described herein and contained by each subgroup may notbe all-inclusive, one of skill in the art can easily determine, byknowing the physicochamical properties of the ingredient, which subgroupcontains other ingredients compatible to that ingredient. Alternatively,if the properties of the new ingredient are not known, routineexperimentation can be used to determine the preferred subgroup. Forexample, each subgroup containing particular media ingredients asdefined according to the present invention, can be supplemented with thenew ingredient. The new ingredient will be compatible with a particularsubgroup when the resultant concentrated solution is soluble and stable.By comparing experimental media preparations to control media not madeaccording to the media concentrate technology, one of skill may furtherdetermine the preferred subgroup. Growth characteristics of cells,formation of toxic products, and shelf-life of the media may be used asindicators of compatible ingredients.

Particular ingredients that have unique physicochemical properties may,in some situations, not be compatible with the ingredients of anysubgroup. In such a case, it may be necessary to maintain this componentas a separate subgroup. Such ingredients may be added to the diluent inan appropriate concentration so that a desired 1× culture media(containing this ingredient) is produced when sufficient amounts of theconcentrated subgroups are admixed with the diluent. Alternatively, theincompatible ingredient can be added as a separate concentratedsolution. Thus, the present invention envisions the development ofadditional subgroups, other than those defined in the present invention,which stabilize and solubilize additional ingredients which may not becompatible with any subgroups defined herein.

It will be apparent from the list of ingredients designated for eachsubgroup described below that a number of ingredients can be included inmore than one subgroup. When a particular ingredient is designated ascompatible with two or more subgroups, that ingredient can be includedin one or more of these subgroups at various concentrations, as long asthe total amount of that ingredient conforms to the final mediaformulation. The only limitation is that the amount of ingredient addeddoes not adversely affect solubility or stability of the otheringredients in the particular subgroup. The number and types ofsubgroups necessary to prepare different media may vary according tomedia complexity and/or the physicochemical properties of theingredients. The duplication of ingredients in more than one subgroupthus provides one of ordinary skill in the art with a versatileclassification scheme to prepare any media.

C. Acid Soluble Subgroup

Media ingredients placed in the acid soluble subgroup comprise all aminoacids (except glutamine), thiamine, ascorbic acid, ferric compounds,ferrous compounds, purines, glutathione and monobasic sodium phosphates.These components are soluble in acid (pH of about 0 to 1.0) and are verystable under these conditions.

Both D and/or L-amino acids may be included in the acid-soluble subgroupof the present invention. Typically, culture medium contain the L-aminoacids. As required by the desired media formulation, one or more aminoacids in any combination may be included in the acid-soluble subgroup,with the exception of glutamine. Glutamine is specifically included inthe glutamine-containing subgroup. Thus, the amino acids that may beincluded in the acid-soluble subgroup of the present invention,depending on the requirements of the desired culture media, includeglycine, alanine, valine, leucine, isoleucine, serine, threonine,cysteine, cystine, methionine, asparagine, aspartic acid, glutamic acid,lysine, arginine, phenylalanine, tyrosine, proline, hydroxyproline,histidine, and tryptophan. Derivatives of amino acids may also beincluded in the acid-soluble group provided that the derivative iscompatible with the other ingredients of this subgroup and with themaintenance or proliferation of the particular cell culture.

Other ingredients which may be included in the acid-soluble subgroup ofthe present invention include thiamine (vitamin B₁), ascorbic acid(vitamin C), purines (guanine and adenine) and glutathione. Derivativesof such compounds are well-known and may be included in the acid-solublegroup, provided that the physicochemical properties have not beenchanged to the extent that the derivative is no longer compatible withthe other ingredients included in this subgroup and with the maintenanceor proliferation of the particular cell culture.

Ferric and ferrous compounds, if required by the media formulation, maybe placed into this subgroup as well. Such ferric and ferrous compounds,that are stable and soluble in the presence of the other ingredientsincluded in this subgroup, can easily be determined by one of skill inthe art. Examples of such ferrous or ferric compounds include, but arenot limited to, Fe(NO₃)₃, FeSO₄, and FeCl₃.

Any monobasic phosphate which is a component of the desired mediaformulation can be contained within the acid-soluble subgroup. Examplesof typical phosphates include, but are not limited to, monobasicphosphates such as NaH₂ PO₄ and KH₂ PO₄.

The acid-soluble subgroup of the present invention may contain variouscombinations of the abovenoted compatible ingredients, as required bythe particular culture media. Thus, according to the classificationscheme of the present invention, the ingredients found in the desiredculture media are selected which are compatible in an acid solution.Once divided in this manner, these ingredients, typically in dry powderform (dehydrated), are dissolved in an acidic solution with a pH ofabout 0 to 1.0. Preferably, the resulting solution is concentrated (10×to 100×). That is, the ingredients in the acid solution are moreconcentrated then the same ingredients found in a 1× media formulation.

As will be apparent, the concentrated acid-soluble subgroup may varyslightly in composition, osmolarity and pH from preparation topreparation of the same media. Furthermore, the acid-soluble subgroupwill also differ from one media to the next. This is true for the othersubgroups as well. The present invention provides the means to produce astable preparation containing acid-soluble media ingredients. Such apreparation can be maintained as a dry powder mix of ingredients butpreferably are maintained as a concentrated solution. Typically, theconcentrated solution comprising the acid soluble subgroup is sterilizedby one of a number of well-known techniques.

D. Glutamine Containing Subgroup

Glutamine is considered an unstable component in liquid media (Tritsch,G. L. et al., Experimental Cell Research 28:360-364 (1962); Ozturk, S.S. et al., Biotechnol. Prog. 6:121-128 (1990)). In solution, glutaminedecomposes to pyrrolidone carboxylic acid and ammonia. The advent ofmedia concentrate technology according to the present invention providesa procedure for the stabilization of glutamine. Thus, cell culture mediathat contains glutamine can be made by preparing, in addition to othersubgroups, a glutamine-containing subgroup.

According to the invention, glutamine (both D and L-forms) can bestabilized in solution by the addition of one or more divalent cations.Any divalent cation, such as Ca⁺², Mg⁺², Mn⁺², Z⁺² and Cu⁺² can be usedto stabilize glutamine. A number of readily available salts including,but not limited to, MgCl₂, CaCl₂, Ca(NO₃)₂, and MgSO₄ may supply thesedivalent cations. Thus, any compatible calcium salt, magnesium salt,and/or manganese salt may be included in this subgroup. Such salts aretypical ingredients of almost any media. Therefore, the divalent saltsof the media formulation should be included in this subgroup tostabilize glutamine. In situations where divalent cations are generallynot ingredients of the desired culture media, divalent cations that donot adversely affect the growth or the maintenance of the cells to begrown should be added to stabilize glutamine in the glutamine-containingsubgroups.

The only limitation, however, with regard to the glutamine-containingsubgroup of the invention is that potassium salts such as potassiumchloride cannot be included in this group. Sodium and magnesium ionsdecrease the solubility of potassium ions. Additionally, metal salts ofphosphate cannot be put into this group since Ca²⁺ forms an insolublecomplex with phosphate, removing Ca²⁺ from the solution.

The amount of glutamine as required by the particular media is mixedwith a sufficient amount of a divalent cation to stabilize glutamine.The ratio of glutamine to divalent cations in this subgroup ispreferably about 2:1 (weight to weight) but may range from 2:1 to 2.9:1.The pH of such a solution is also important for stabilizing glutamine.The preferred pH may range from 4.0 to 6.0. The most preferred pH of theglutamine-divalent cation solution is about 5.5. Basic pHs tend to lowerglutamine stability, and thus, if necessary, pH may be adjusted with anacid which is compatible with the glutamine containing subgroup, e.g.HCl.

When the ratio of glutamine to divalent cation is about 2:1, glutamineforms a stable soluble complex. This complex does not favor thebreakdown of glutamine to pyrrolidone carboxylic acid and ammonia. Theglutamine metal complex thus provides a means for supplying thisessential nutrient in a stable, soluble liquid form.

The ingredients contained in the glutamine-containing subgroup maycomprise glutamine, calcium salts, and/or magnesium salts. D-Capantothenate may also be included in the glutamine containing subgroup,as well as sodium chloride.

As will be evident, each culture medium prepared according to thepresent invention may have a preferred osmolarity range. Osmolarity willchange, as will the final pH of the culture media, with the amount ofacid/base used for solubilization of ingredients in each subgroup. Oneof skill in the art can easily determine the amount of salt, e.g.,sodium chloride, necessary to obtain the preferred final osmolarity ofthe desired culture media by taking into consideration the final pH andvolume of each concentrated subgroup required to prepare the mediaformulation. Such parameters (pH of each subgroup and total salt content) can be easily determined by routine experimentation. For example, thepH of the ingredient containing subgroups can be measured by well-knowntechniques. The osmolarity of the 1× media formulation, prepared bymixing an appropriate volume of these concentrated subgroups allows oneof ordinary skill in the art to determine the amount of sodium chlorideneeded, if any, to adjust the final osmolarity of the culture media.This amount of sodium chloride may be added to the glutamine-containingsubgroup to achieve the proper final osmolarity. Most 1× mediaformulations have broad range requirements for final osmolarity and pHand therefore slight variations in subgroup pH and salt concentrationare not critical.

E. Weak Acid and Base Soluble Subgroup

The media ingredients of the weak acid-weak base soluble subgroup(referred to herein as a weak acid-base soluble subgroup) consist ofsugars, deoxyribose, ribose, nucleotides, water-soluble vitamins (exceptfor thiamine, ascorbic acid and Ca-pantothenate) riboflavin, salts,e.g., potassiumchloride and sodium chloride, trace metals, lipids,acetate salts, phosphate salts, HEPES, phenol red, pyruvate salts, andbuffers.

The pH of the weak acid-base subgroup may range from about 4.0-9.0.Dibasic sodium phosphate and some monobasic sodium phosphate may also begrouped here. The combination of dibasic and monobasic sodium phosphateprovides a buffer system for this concentrated subgroup. Adjustment ofpH to the solution of this subgroup is preferably made by the additionof sodium hydroxide or hydrochloric acid, as appropriate, but notpotassium hydroxide.

The ingredients found in this subgroup are determined, as with all othersubgroups, by the media composition. Thus, any combination of the aboveingredients may be included in the weak acid-base soluble group.

Sugars that are included in this subgroup include any sugar that can beused as a media ingredient and is compatible in combination with one ormore of the above ingredients. Such sugar compounds include glucose,galactose, maltose, mannose, etc., and derivatives thereof.

Sugars in this subgroup may also serve as a vehicle for solubilizinglipids, if lipids are included in this subgroup. Lipids can besolubilized in solution after being desiccated onto a sugar or saltmolecule. Briefly, lipids are solubilized in a small volume of ethanoland then slowly dripped onto a dry powder of sugar/salt. The mixture isdried for 3 hours and then ball milled. The sugar-lipid or salt-lipidcomposition can then be prepared as a concentrate comprising the otheringredients of the weak acid-base subgroup.

Any lipid and/or fat soluble ingredient including, but not limited to,linoleic acid, lipoic acid, cholesterol, calciferol, vitamin A,2-mercaptoethanol, tween 80, menadione and vitamin E can be desiccatedonto a salt or sugar and thus included in this subgroup.

Any deoxyribose, ribose or nucleotide may be included in the weak acidand base soluble subgroup according to the present invention. Thesecompounds and their derivatives are well-known. Examples includeadenosine and adenosinetriphosphate. Again, the only limitation is thatthe ingredient must be compatible with the other ingredients found inthis subgroup.

Vitamins in the weak acid-base subgroup may include all water-solublevitamins and their derivatives except thiamine, ascorbic acid andCa-pantothenate. Such water-soluble vitamins that maybe included in theweak acid-base soluble group include folio acid, biotin, pyridoxine,nicotinic acid, riboflavin, and the like.

Trace metals, used in media formulations, are also included in thissubgroup. Examples of trace metals include CuSO₄, Na₂ SeO₃, ZnSO₄, andCoCl₂. Trace amounts of other metals may also be included in this group.The selection of appropriate trace metals and the amounts employed maybe readily determined by one of ordinary skill in the art.

Potassium chloride, dibasic sodium phosphate, sodium acetate, phenolred, sodium chloride, HEPES, and sodium pyruvate are also compatibleingredients in the weak acid-base subgroup. Derivatives of any of thecompounds can also be employed in this group as long as such derivativesare compatible.

Riboflavin may also be placed in this group. Riboflavin is aphotosensitizer and is the main nutrient responsible for the generationof hydrogen peroxides through the interaction with histidine,methionine, tyrosine and tryptophan (Griffin, F. M., et al., CancerResearch 91:2241-2248 (1981); Wang, R. J., et al., In Vitro 14:715-722(1978)). By separating riboflavin from the amino acids, a stableconcentrated subgroup is created.

A component that lends stability to liquid media subgroup comprisingriboflavin is pyruvate. Pyruvate interacts with hydrogen peroxide togive acetic acid, carbon dioxide and water (Weil, L., Gordon, W. G., andBuchert, A. R., Arch. Biochem. Biophys. 33:90-109 (1951)). Sincehydrogen peroxide is deleterious to cultured cells, membrane proteins,lipids and enzymes, controlling its build-up may be critical in aparticular media. Thus, even if the desired media does not requirepyruvate, the addition of pyruvate helps to increase the stability ofthe weak acid-base soluble subgroup and the shelf-life of the mediaprepared from this subgroup.

F. Alcohol Soluble Subgroup

Media formulations that contain cholesterol, steroids, tween 80,fat-soluble vitamins or other hydrophobic components may be concentratedinto an alcohol-soluble group. Other hydrophobic compounds may beincluded in the alcohol-soluble group are well known to those of skillin the art.

Any combination of these components can be solubilized in a small volumeof ethanol or propylene glycol. Once solubilized, these ingredients arethen microfluidized by well known techniques to form small lipidvesicles (Lidgate et al., Bio Pharm 2(9): 28-33 (1989)). Briefly, themicrofluidizer (Model 110 Microfluidizer) produces emulsions bycombining shear, turbulence, and cavitation forces. Each cycle throughthe microfluidizer decreases the emulsion's mean droplet size ultimatelyproducing submicron particles that are very stable. Water solublecomponents can also be trapped into these vesicles and then delivered tothe culture media.

Fat soluble vitamins including vitamin A, D, E and K may also beincluded in this subgroup, as well as their derivatives. Steroids andtheir derivatives including cortisol, progesterone, estrogen,testosterone etc. are also included within the alcohol-soluble subgroup.

Fatty acids and lipids of the media formulation may be divided into thissubgroup. Any fatty acid such as linoleic acid, oleic acid, arachidonicacid etc. fall into this classification. As will be appreciated, anylipid may also be included. Examples of lipids are lecithin,sphingomyelin and phospholipids. Lipids (oils) are usually necessaryingredients in this subgroup since microfluidization requires lipids(oils) to prepare lipid vesicles. In situations where the mediaformulation requires lipids but not the hydrophobic ingredients of thissubgroup, it may be unnecessary to prepare an alcohol-soluble subgroupsince such lipids can be included in the weak acid-base soluble group.In the weak acid-base soluble subgroup, lipids are desiccated ontosugars and/or salts.

Other media components that may be included in the alcohol-soluble groupof the present invention may include tween 80 and 2-mercaptoethanol.

G. Alkali-Soluble Subgroups

For media components that require an alkaline environment forsolubilization (pH 9.0), an alkali soluble subgroup is employed. Thissubgroup consists of the pyrimidines and purines that are stable andsoluble at alkaline pH. These components, when dissolved in NaOH, formsodium salts which are soluble in an aqueous environment.

Pyrimidines and their derivatives that are compatible in this alkalisubgroup include uracil and thymine. Xanthine and hypoxanthine areexamples of purines that may also be include into the alkali solublesubgroup of the present invention. Other ingredients that are stable andsoluble in this alkali environment are known by or easily determined bythose of skill in the art.

H. Supplement-Containing Subgroup

As previously stated, typical medium formulations comprise amino acids,salts, vitamins, trace metals, sugars, lipids and nucleic acids.However, media are often supplemented with proteins, antibiotics orundefined broths/sera. When such a supplement is required for aparticular medium, the supplement can be used directly (e.g., serum) ormade into a concentrate (protein solutions such as insulin, transferrinand growth factors). The supplement-containing subgroup can then becombined with the other concentrated subgroups to produce the desiredmedia formulation.

A number of components that are included in the final media formulation,but not classified as a compatible ingredient in one of theaforementioned subgroups, may be added as one or more separatesupplements. Thus, the present invention envisions the preparation ofone or more supplement-containing subgroups that are not compatible inthe above-noted subgroups. Such components that fall into the supplementsubgroup include serum (fetal, horse, calf, etc.), yeastolateultrafiltrate, tryptose phosphate broth, protein concentrates (insulin,transferrin, growth factors, hormones, albumin, etc.), antibiotics(gentamicin, penicillin, streptomycin, amphotericin B, etc.), whole eggultrafiltrate, attachment factors (fibronectin, vitronectin, collagen,laminin, etc.) and NaHCO₃ liquid concentrate (for example, 25×).Depending on the final media formulation, one of ordinary skill in theart may select appropriate supplements which are compatible.

I. Kits

The subgroups of compatible ingredients of the invention are ideallysuited for preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, test tubes, bottles, drums, and the like.Each of said container means comprises one of the individual subgroupsof compatible ingredients as defined above. Such subgroups may behydrated or dehydrated but are typically a concentrated liquid solution.Such solutions may, according to the invention, be sterile.

A first container means may, for example, comprise any combination ofmedia ingredients that are compatible in the acid-soluble subgroup.Compatible ingredients or any combination thereof that are included inthe weak acid-base soluble group may be contained in a second containermeans. A third and fourth container means may comprise any combinationof ingredients included in the alkali-soluble subgroup andalcohol-soluble subgroup, respectively. A fifth container means maycomprise any combination of ingredients included in theglutamine-containing subgroup as long as divalent cations are includedto stabilize the glutamine. A sixth container means may compriseingredients listed in the supplement-containing subgroup. Otheringredients that are not compatible in the defined subgroups of theinvention may be contained in one or more further container means toavoid mixing of incompatible components.

The number and types of subgroup containing container means comprising akit for making a cell culture media may vary depending on the type ofmedia to be prepared. Typically, the kit will contain the respectivecontainer means containing the subgroups necessary to make a particularmedia. However, additional subgroup containing container means maybeincluded in the kit of the invention so that different media can beprepared by mixing different amounts of various subgroups to makedifferent media formulations.

J. Mixing Concentrated Subgroups to Prepare a Culture Media

The media concentrate technology of the present invention provides ameans to prepare one or more concentrated solutions of compatibleingredients by dividing these ingredients into the subgroups definedabove. These subgroups, once prepared as concentrated solutions (greaterthan 1× ), can be combined with a diluent to prepare the desired culturemedia. The amount of concentrated solution and amount of diluent neededmay vary depending on the concentration of each subgroup, the number ofsubgroups and the desired concentration of the final media. One ofordinary skill in the art can easily determine a sufficient volume of adiluent and a sufficient volume of these concentrated solutions toprepare the desired media.

The pH of the resulting culture media can be adjusted by addition ofacid or base. The media, however, may not require any adjustment,especially if the pH of each subgroup concentrate is adjusted so thatthe pH of the prepared media is in the desired range. Osmolarity of themedia can also be adjusted after mixing the concentrated solutions witha diluent. Typically, the desired osmolarity may be predetermined andadjustments in the salt concentration of the concentrated solutions maybe made to prepare a final media with the desired osmolarity.

K. Uses

The present invention provides concentrates that can easily be mixed insufficient amounts with a diluent to prepare a culture media. Mixingthese concentrated subgroups can be done on a batch or continuous basis.In a batch system, appropriate amounts of the subgroup concentrates canbe admixed with a diluent to prepare a single quantity of media. In acontinuous system, sufficient amounts of each subgroup concentrate arecontinuously admixed with sufficient amounts of a diluent in a mixingchamber, while the culture media is continually removed. The obviousadvantage of the continuous system is that a single reactor (mixingchamber) can be used to prepare media for a number of culture vessels orfermentation tanks. In addition, the continuous preparation of media inthis manner can be accomplished in a closed sterile system.

According to the present invention, appropriate media concentrates canbe prepared for any media and in any quantity. Such concentrates canthus be prepared and supplied commercially or made and used for in-houseresearch. Customers that obtain prepared concentrates may prepare mediain a more cost-effective manner. For example, the life of mixing tankswill be increased since certain corrosive dry powders need not be usedto prepare media. Also, fewer man-hours may be needed to prepare thedesired culture media.

Having now fully described the present invention, the same will be moreclearly understood by reference to certain specific examples which areincluded herewith for purposes of illustration only, and not intended tobe limiting of the invention, unless specified.

EXAMPLE I Stabilization of Glutamine with Divalent Cations

L-Glutamine was admixed in a solution with varying amounts of calciumnitrate and magnesium sulfate at a pH of 5.5. FIG. 1 shows that when theratio of L-glutamine to divalent cations is significantly above 2.5:1,the stability of glutamine decreased. L-Glutamine was assayed byprecolumn derivatization with O-phthaldialdehyde and then detected usinghigh performance liquid chromatography (Rajendra, W. J. Liquid Chromat,10(5):941-955 (1987)).

At a 2.5:1 glutamine to divalent cation ratio, glutamine remained stablein solution for 24 weeks. Cell culture media containing glutamine alsocontains divalent cations but due to the interaction of divalent cationswith other amino acids present in the culture media, glutamine was notstabilized in 1× media formulation and begins to slowly degrade at 4° C.with only 65-70% remaining after three weeks. (Sigma Cell CultureReagents Catalogue (1991) pg. 202.)

EXAMPLE II Preparation of RPMI-1640 Media

RPMI-1640 media is a media commonly used to grow mammalian cells inculture. The pH of this media is about 7.0 and the osmolarity can rangefrom 265 to 300.

RPMI-1640 contains amino acids, including L-glutamine, various salts,vitamins, sugars, etc. The ingredients found in this media are describedin GIBCO/BRL Catalogue and Reference Guide, pg. 118, 1990.

The ingredients of RPMI-1640 media were divided into a number ofcompatible subgroups. Once the dry powder compatible ingredients weremixed, a concentrated solution of each subgroup was prepared. Anappropriate volume of each of the prepared concentrated subgroups wasthen used to prepare a 1× RPMI-1640 media formulation.

Table 1 shows the ingredients of the RPMI-1640 media that were dividedinto the acid soluble subgroup. The concentrations correspond to a 1×formulation in 1 liter of solution. To make a 100×formulation of theacid soluble subgroup, the amount of each ingredient shown in Table 1was added together to prepare a dry powder mix (DPM) Of acid solubleingredients. For example, 0.2 g of L-arginine, 0.05 g L-asparagine, 0.02g L-aspartic acid, etc. were added to prepare a dry powder mix totaling0.69 grams.

                  TABLE 1                                                         ______________________________________                                        Acid-Soluble Subgroup                                                         for RPMI-1640 Media                                                                       Molecular                                                         Component   Weight       g/l     μM                                        ______________________________________                                        L-Arginine  174          .200    1,149.43                                     L-Asparagine                                                                              132          .050    378.78                                       L-Aspartic Acid                                                                           133          .020    150.37                                       L-Cystine.2HCl                                                                            313          .06515  208.15                                       L-Glutamic Acid                                                                           147          .020    136.05                                       Glycine      75          .010    133.33                                       L-Histidine 155          .015    96.77                                        L-Hydroxyproline                                                                          131          .020    152.67                                       L-Isoleucine                                                                              131          .050    381.67                                       L-Leucine   131          .050    381.67                                       L-Lysine.HCl                                                                              183          .040    218.58                                       L-Methionine                                                                              149          .015    100.67                                       L-Phenylalanine                                                                           165          .015    89.82                                        L-Proline   115          .020    173.91                                       L-Serine    105          .030    285.71                                       L-Threonine 119          .020    168.07                                       L-Tryptophan                                                                              204          .005    24.51                                        L-Tyrosine  181          .02086  115.32                                       L-Valine    117          .020    170.94                                       Thiamine.HCl                                                                              337          .001    2.967                                        ______________________________________                                    

As shown in Table 2, the acid soluble compatible ingredients were mixedwith 8.8 ml of water and then HCl was added to give a final pH of 0.85.This required about 1.2 ml of 5N HCl. The resulting solution was a100×formulation of the acid soluble subgroup for RPMI-1640 media.

Based on the 1640 media formulation, two additional subgroups wereprepared: a glutamine-containing subgroup and a weak acid-base solublesubgroup. Table 3 shows the ingredients added to make a dry powder mixof the glutamine-containing subgroup for RPMI-1640 media. The mixture ofingredients totaling 6.4 g was mixed with about 17.5 ml water to preparea 20 ml 50× concentrate with a final pH of about 5.5.

Table 4 shows the amount of ingredients needed to make 3.25 g of drypowder mix (DPM) for the weak acid-base soluble group. About 19.5 mlwater was added to the weak acid-base ingredients to prepare a 20 mlsolution with a pH of about 8.3. All ingredients in this subgroup werewater soluble and therefore did not require desiccation onto a salt orsugar.

Reconstitution of the above-described acid-soluble subgroup,glutamine-containing subgroup and weak acid-base soluble subgroup wasaccomplished as shown in Table 2 to prepare a 1× RPMI-1640 mediaformulation. NaHCO₃ was added as a separate supplement. The osmolarityof the media was 276 mOm before filter sterilization. Alternatively, aconcentrated solution of NaHCO₃ can also be used rather than adding drypowder NaHCO₃ to the reconstituted media.

                  TABLE 2                                                         ______________________________________                                        Procedure for Preparation of RPMI-1640 Subgroups                              1.     Weak Acid-Base Soluble Subgroup (50X Concentrate)                             Quantity sufficient to make 1 liter                                           19.5 ml water                                                                  3.25 g DPM                                                                   20.0 ml Total Volume  Final pH = 8.28                                         Special Instructions: Add DPM slowly to water to avoid                        solidification of components. Use moderate stirring until                     dissolved (about 15-20 minutes).                                       2.     Acid Soluble Subgroup (100X Concentrate)                                      Quantity sufficient to make 1 liter                                           8.8 ml water                                                                  0.69 g DPM                                                                    1.20 ml 5N HCl                                                                10.0 ml Total Volume  Final pH = 0.85                                         Special Instructions: Add 5N HCl slowly to the stirred                        solution.                                                              3.     Glutamine-Containing Subgroup (50X Concentrate)                               Quantity sufficient to make 1 liter                                           17.5 ml water                                                                 6.4 g DPM                                                                     20.0 ml Total Volume  Final pH = 5.50                                         Special Instructions: This is an endothermic process. The                     temperature of the solution will drop about 3° C. with the             addition of components. Use moderate stirring for about                       10-15 minutes or until all components are dissolved.                   Procedure for Preparing a 1X RPMI-1640 Media Formulation:                     1.    Add subgroup concentrates to water in the following order:               A. 950 ml water           pH 5.98                                             B.  20 ml Glutamine-Containing Subgroup                                                                 pH 5.61                                             C.  20 ml Weak Acid-Base Soluble Subgroup                                                               pH 8.20                                             D.  10 ml Acid Soluble Subgroup                                                                         pH 4.91                                             E.  2.0 g NaHCO.sub.3     pH 7.00                                            Osmolarity before filtration: 276 m OSm                                       ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Glutamine-Containing Subgroup                                                 for RPMI-1640 Media                                                                          Molecular                                                      Component      Weight     g/l    μM                                        ______________________________________                                        Ca(NO.sub.3).sub.2                                                                           164.10     .0695  423.52                                       MgCl.sub.2     95.23      .0386  405.75                                       NaCl           58.44      6.000  102,669.4                                    L-Glutamine    146.00     .300   2,054.79                                     D-Ca Pantothenate                                                                            476.00     .0025  0.525                                        ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Weak Acid-Base Soluble Subgroup                                               for RPMI-1640 Media                                                                        Molecular                                                        Component    Weight     g/l     μM                                         ______________________________________                                        Biotin       244.00     .0002   .8196                                         Choline Chloride                                                                           140.00     .0030   21.4300                                       Folic Acid   441.00     .0010   2.2670                                        i-inositol   180.00     .0350   194.4400                                      Niacinamide  122.00     .0010   8.1960                                        Para-Aminobenzoic                                                                          137.13     .0010   7.2900                                        Acid                                                                          Pyridoxine-HCl                                                                             206.00     .0010   4.8540                                        Riboflavin   376.00     .0002   .5320                                         Vitamin B12  1355.00    .000005 .00369                                        Glucose      180.16     2.0000  11,101.2400                                   Glutathione  307.33     .0010   3.2500                                        Phenol Red   376.36     .0050   13.2900                                       Na.sub.2 HPO.sub.4                                                                         141.96     .8000   5635.3900                                     KCl          74.56      .4000   5364.8100                                     ______________________________________                                    

EXAMPLE III Reconstitution of RPMI-1640 Medium Concentrated SubgroupsUsing a Continuous Feed Mixing Chamber

A reconstitution experiment was carried out using RPMI-1640 mediaconcentrate subgroups in the 15L Biospin bioreactor. Three mediasubgroups at 50× per subgroup and two 25× NaHCO₃ subgroup werecontinually pumped into the bioreactor at a rate of 250 ml/hour.Deionized distilled water was pumped into the reactor at a rate of 12.5Lper hour. The perfused concentrates were continually admixed in themixing chamber. Complete 1× medium formulation was removed from theBiospin bioreactor at a rate of 12.5L per hour. The pH of the medium wasmonitored both internally and externally. Medium parameters (pH,osmolarity, amino acids, glucose, glutamine) were measured throughoutthe perfusion. The results of the study indicate that RPMI-1640concentrated subgroups can be reconstituted in the Biospin bioreactorand the resulting 1× medium formulation parameters show significantcorrelation with the RPMI-1640 formulation (FIGS. 2,3 and Table 5).

                  TABLE 5                                                         ______________________________________                                        Concentrate Reconstitution of RPMI 1640 Trial 1                               HPLC Assay Results                                                                     Medium  Conc.   Hour  Hour  Hour  Hour                               Amino Acid                                                                             Theo.   Std     1     3     5     6.5                                ______________________________________                                        Aspartate                                                                              20.00   20.30   21.00 20.93 20.77 19.47                              Glutamate                                                                              20.00   20.84   23.91 22.46 21.05 19.95                              Hydroxyproline                                                                         20.00   25.92   24.98 26.93 25.39 24.61                              Asparginine                                                                            50.00   41.60   44.84 45.96 45.70 44.34                              Arginine 200.00  192.03  199.53                                                                              197.26                                                                              198.54                                                                              195.83                             Threonine                                                                              20.00   33.04   40.01 34.91 35.63 30.51                              Proline  20.00   32.78   36.93 31.11 32.05 32.34                              Tyrosine 20.00   25.84   26.63 26.77 24.81 19.11                              Valine   20.00   22.15   22.90 22.66 21.83 18.40                              Methionine                                                                             15.00   16.12   17.18 18.06 17.70 14.00                              Cysteine 50.00   35.64   39.51 39.09 43.04 34.86                              Isoleucine                                                                             50.00   56.71   53.72 51.85 51.38 49.54                              Leucine  50.00   54.04   52.97 52.40 50.74 50.25                              Phenylalanine                                                                          15.00   16.59   19.76 18.88 19.61 17.73                              Tryptophan                                                                             5.00    4.66    7.67  7.62  7.99  6.47                               Lysine   40.00   28.41   32.37 29.00 32.87 30.05                              Histidine                                                                              15.000  15.000  14.414                                                                              13.899                                                                              12.884                                                                              10.540                             Serine   30.000  30.000  31.030                                                                              30.399                                                                              30.745                                                                              22.250                             Glycine  10.000  10.000  11.495                                                                              13.588                                                                              12.677                                                                              9.172                              ______________________________________                                    

EXAMPLE IV Preparation of DMEM Media

DMEM media (Dulbecco's Modified Eagle's Medium (GIBCO/BRL Catalogue andReference Guide, pg. 96, 1990; Dulbecco, R. et al. Virology 8:396(1959); and Smith, J. D. et al. Virology 12:185 (1960) was prepared bydividing the ingredients of this media into an acid-soluble subgroup, aglutamine-containing subgroup and a weak acid-base soluble subgroup. Theingredients of DMEM media were divided and prepared as a concentratedsubgroups as shown in Tables 6, 7, 8 and 9. A procedure similar to thatdescribed in Example II was used.

Table 9 also shows the procedure used for mixing and diluting the DMEMsubgroups to make a 1× formulation of DMEM. NaOH was added to the 1×media to adjust the final pH to 7.3. NaHCO₃ was added as a bufferingagent.

                  TABLE 6                                                         ______________________________________                                        Acid Soluble Subgroup for DMEM Media                                          Components    MW          g/L      uM                                         ______________________________________                                        L-Arginine.HCl                                                                              210.66      0.084    398.7                                      L-Cystine.2HCL                                                                              313.22      0.06257  199.8                                      L-Glycine     75.0        0.030    400.0                                      L-Histidine.HCl.H.sub.2 O                                                                   209.46      0.042    200.5                                      L-Isoleucine  131.0       0.105    801.5                                      L-Leucine     131.0       0.105    801.5                                      L-Lysine.HCl  182.46      0.146    800.2                                      L-Methionine  149.0       0.030    201.3                                      L-Phenylalanine                                                                             165.0       0.066    400.0                                      L-Serine      105.0       0.042    400.0                                      L-Threonine   119.0       0.095    798.3                                      L-Tryptophan  204.0       0.016    78.4                                       L-Tyrosine    181.0       0.072    397.8                                      L-Valine      117.0       0.094    803.4                                      NaH.sub.2 PO.sub.4.H.sub.2 O                                                                137.98      0.125    905.9                                      Thiamine.HCl  337.28      0.004    11.9                                       FeC13.6H.sub.2 O                                                                            270.22      0.000054 0.2                                        ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Glutamine-Containing Subgroup                                                 for DMEM Media                                                                Components   MW          g/L     uM                                           ______________________________________                                        CaCl.sub.2 (anhyd)                                                                         110.99      0.200   1802.0                                       NaCl         58.45       5.400   92386.7                                      MgCl2(anhyd) 95.23       0.07726 811.3                                        D-Ca Pantothenate                                                                          476.53      0.004   8.4                                          L-Glutamine  146.15      0.584   3995.9                                       ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Weak Acid-Base Soluble Solution                                               for DMEM Media                                                                Components   MW          g/l     uM                                           ______________________________________                                        NaCl         58.45       0.400   6843.5                                       Glucose      180.16      4.500   24977.8                                      Na-Pyruvate  111.05      0.110   990.5                                        Choline Chloride                                                                           140.0       0.004   28.6                                         Folic Acid   441.0       0.004   9.1                                          Inositol     180.0       0.0072  40.0                                         Niacinamide  122.0       0.004   32.8                                         Pyridoxal.HCl                                                                              203.62      0.004   19.6                                         Riboflavin   376.0       0.0004  1.1                                          KCl          74.56       0.400   5364.8                                       ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Procedure for Preparation of DMEM Subgroups                                   1.  DMEM High Glucose                                                             Glutamine-Containing Subgroup                                                 18.1 ml H2O                                                                   6.3 g DPM                                                                     20.0 ml Total Volume                                                                              pH 5.50 5     0   X                                       Concentrate                                                               2.  DMEM High Gludose                                                             Weak Acid-Base Soluble Subgroup                                               17.0 ml H2O                                                                   5.4 g DPM                                                                     0.7 ml 0.1N NaOH                                                              20.0 ml Total Volume                                                                              pH 6.60 5     0   X                                       Concentrate                                                               3.  DMEM High Glucose                                                             Acid Soluble Subgroup                                                         18.5 ml H2O                                                                   1.1 g DPM                                                                     1.5 ml 5N HCl                                                                 20.0 ml Total Volume                                                                              pH 0.80 5     0   X                                       Concentrate                                                               Procedure for Preparing a 1X DMEM Media                                       Formulation                                                                    900.0 ml H2O                                                                  20.0 ml Glutamine-Containing Subgroup                                         20.0 ml Weak Acid-Base Subgroup                                               20.0 ml Acid Soluble Subgroup                                                  3.7 g NaHCO3                                                                ADD 5N NaOH to brinq pH to 7.30                                               ADD H2O to bring volume to 1000.0 ml                                          1000.0 ml Total Volume                                                                            pH 7.30 1X Formulation                                    Osmolarity will be 315-340 mOsm. Osmolarity will                              change vith the amount of acid/base used for the                              solubilization of components in each subgroup.                                If the osmolarity falls above the desired limits                              an adjustment in the NaCl concentration can be                                made to lower the osmolarity.                                                 ______________________________________                                    

EXAMPLE V Preparation of HAM's F-12 Medium

HAM's F-12 medium (Ham, R. G., Proc. Natl. Acad. Sci. 53:288 (1965)) isprepared by dividing the ingredients of this media into an acid-solublesubgroup, a glutamine-containing subgroup, and a weak acid/basesubgroup. The compositions of the respective subgroups are set out inTables 10, 11 and 12. A procedure similar to that described in ExampleII may be used to prepare the media concentrates.

                  TABLE 10                                                        ______________________________________                                        ACID SOLUBLE SUBGROUP FOR HAM'S F-12 MEDIUM                                   COMPONENT          mg/L                                                       ______________________________________                                        L-ALANINE          8.90                                                       L-ARGININE.HCL     211.00                                                     L-ASPARAGINE.H.sub.2 O                                                                           15.01                                                      L-ASPARTIC ACID    13.30                                                      L-CYSTEINE. HCL.H.sub.2 O                                                                        35.12                                                      L-GLUTAMIC ACID    14.70                                                      GLYCINE            7.50                                                       L-HISTIDINE.HCL.H.sub.2 O                                                                        20.96                                                      L-ISOLEUCINE       3.94                                                       L-LEUCINE          13.10                                                      L-LYSINE.HCL       36.50                                                      L-METHIONINE       4.48                                                       L-PHENYLALANINE    4.96                                                       L-PROLINE          34.50                                                      L-SERINE           10.50                                                      L-THREONINE        11.90                                                      L-TRYPTHPHAN       2.04                                                       L-TYROSINE         5.40                                                       L-VALINE           11.70                                                      THIAMINE.HCL       0.34                                                       FeSO.sub.4.7H.sub.2 O                                                                            0.834                                                      ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        GLUTAMINE CONTAINING SUBGROUP                                                 FROM HAM'S F-12 MEDIUM                                                        COMPONENT          mg/L                                                       ______________________________________                                        L-GLUTAMINE        146.00                                                     D-CA PANTOTHENATE  0.48                                                       CaCl.sub.2 (anhyd) 33.22                                                      MgCl.sub.2 (anhyd) 57.22                                                      NaCl               6000.00                                                    ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        WEAK ACID/BASE SUBGROUP FOR HAM'S F-12 MEDIUM                                 COMPONENT            mg/L                                                     ______________________________________                                        BIOTIN               0.0073                                                   CHOLINE CHLORIDE     13.9600                                                  FOLIC ACID           1.3000                                                   i-INOSITOL           18.0000                                                  NIACINAMIDE          0.0370                                                   PYRIDOXINE.HCL       0.0620                                                   RIBOFLAVIN           0.0380                                                   VITAMINE B.sub.12    1.3600                                                   D-GLUCOSE            1802.0000                                                HYPOXANTHINE (sodium salt)                                                                         4.7700                                                   LINOLEIC ACID        0.0840                                                   LIPOIC ACID          0.2100                                                   PHENOL RED           1.2000                                                   PUTRESCINE.2HCL      0.1610                                                   SODIUM PYRUVATE      110.0000                                                 THYMIDINE            0.7300                                                   CuSO.sub.4.5H.sub.2 O                                                                              0.0025                                                   KCL                  223.6000                                                 NaCl                 1599.0000                                                Na.sub.2 HPO.sub.4 (anhyd)                                                                         142.0400                                                 ZnSO.sub.4.7H.sub.2 O                                                                              0.8630                                                   ______________________________________                                    

EXAMPLE VI Preparation of DMEM/F-12 Medium

DMEM/F-12 medium (GIBCO/BRL Catalogue and Reference Guide, page 97(1990)) is prepared by dividing the ingredients of this media into anacid-soluble subgroup, a glutamine-containing subgroup, and a weakacid/base subgroup. The compositions of the respective subgroups are setout in Tables 13, 14 and 15. A procedure similar to that described inExample II may be used to prepare the media concentrates.

                  TABLE 13                                                        ______________________________________                                        ACID SOLUBLE SUBGROUP FOR DMEM/F-12 MEDIUM                                    COMPONENT          mg/L                                                       ______________________________________                                        L-ALANINE          4.45                                                       L-ARGININE.HCL     147.50                                                     L-ASPARAGINE.H.sub.2 O                                                                           7.50                                                       L-ASPARTIC ACID    6.65                                                       L-CYSTEINE.HCL.H.sub.2 O                                                                         17.56                                                      L-GLUTAMIC ACID    7.35                                                       GLYCINE            18.75                                                      L-HISTIDINE.HCL.H.sub.2 O                                                                        31.48                                                      L-ISOLEUCINE       54.47                                                      L-LEUCINE          59.05                                                      L-LYSINE.HCL       91.25                                                      L-METHIONINE       17.24                                                      L-PHENYLALANINE    35.48                                                      L-PROLINE          17.25                                                      L-SERINE           26.25                                                      L-THREONINE        53.45                                                      L-TRYPTOPHAN       9.02                                                       L-TYROSINE         38.39                                                      L-VALINE           52.85                                                      THIAMINE.HCL       2.17                                                       FeSO.sub.4.7H.sub.2 O                                                                            0.417                                                      NaH.sub.2 PO.sub.4.H.sub.2 O                                                                     42.50                                                      L-CYSTINE.2HCL     31.29                                                      Fe(NO.sub.3).9H.sub.2 O                                                                          0.05                                                       ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        WEAK ACID/BASE SUBGROUP FOR DMEM/F-12 MEDIUM                                  COMPONENT            mg/L                                                     ______________________________________                                        BIOTIN               0.0035                                                   CHOLINE CHLORIDE     8.9800                                                   FOLIC ACID           2.6500                                                   i-INOSITOL           12.6000                                                  NIACINAMIDE          2.0200                                                   PYRIDOXINE.HCL       0.0310                                                   RIBOFLAVIN           0.2190                                                   VITAMINE B.sub.12    0.0680                                                   D-GLUCOSE            3151.0000                                                HYPOXANTHINE (sodium salt)                                                                         2.3900                                                   LINOLEIC ACID        0.0420                                                   LIPOIC ACID          0.1050                                                   PHENOL RED           8.1000                                                   PUTRESCINE.2HCL      0.0810                                                   SODIUM PYRUVATE      55.0000                                                  THYMIDINE            0.3650                                                   CuSO.sub.4.5H.sub.2 O                                                                              0.0013                                                   KCL                  311.8000                                                 NaCL                 999.5000                                                 Na.sub.2 HPO.sub.4 (anhyd)                                                                         71.02                                                    ZnSO.sub.4.7H.sub.2 O                                                                              0.4320                                                   NaH.sub.2 PO.sub.4.H.sub.2 O                                                                       20.0000                                                  PYRIDOXAL.HCL        2.0000                                                   HEPES                3574.5000                                                ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        GLUTAMINE CONTAINING SUBGROUP                                                 FOR DMEM/F-12 MEDIUM                                                          COMPONENT          mg/L                                                       ______________________________________                                        L-GLUTAMINE        365.00                                                     D-CA PANTOTHENATE  2.24                                                       CaCL.sub.2 (anhyd) 116.60                                                     MgCl.sub.2 (anhyd) 28.64                                                      MgSO.sub.4         48.84                                                      NaCl               6000.00                                                    ______________________________________                                    

EXAMPLE VII Preparation of IMDM Medium

IMDM medium (Dulbecco, R. and Freeman, G., Virology 8:396 (1959); Smith,J. D. et al., Virology 12:185 (1960); Tissue Culture StandardsCommittee, In Vitro 6:2,93; In Vitro 9:6 (1970); Iscove, N. N. andMelchers, F., J. Exp. Med. 147:923) is prepared by dividing theingredients of this media into an acid-soluble soluble subgroup, aglutamine-containing subgroup, and a weak acid/base subgroup. Thecompositions of the respective subgroups are set out in. Tables 16, 17and 18. A procedure similar to that described in Example II may be usedto prepare the media concentrates.

                  TABLE 16                                                        ______________________________________                                        ACID SOLUBLE SUBGROUP FOR IMDM MEDIUM                                         COMPONENT          mg/L                                                       ______________________________________                                        L-ALANINE          25.00                                                      L-ARGININE.HCL     84.00                                                      L-ASPARAGINE.H.sub.2 O                                                                           84.00                                                      L-ASPARTIC ACID    30.00                                                      L-CYSTINE.2HCL     91.24                                                      L-GLUTAMIC ACID    75.00                                                      GLYCINE            30.00                                                      L-HISTIDINE.HCL.H.sub.2 O                                                                        42.00                                                      L-ISOLEUCINE       105.00                                                     L-LEUCINE          105.00                                                     L-LYSINE.HCL       146.00                                                     L-METHIONINE       30.00                                                      L-PHENYLALANINE    66.00                                                      L-PROLINE          40.00                                                      L-SERINE           42.00                                                      L-THREONINE        95.00                                                      L-TRYPTHPHAN       16.00                                                      L-TYROSINE         71.43                                                      L-VALINE           94.00                                                      THIAMINE.HCL       4.00                                                       NaH.sub.2 PO.sub.4.H.sub.2 O                                                                     125.00                                                     ______________________________________                                    

                  TABLE 17                                                        ______________________________________                                        WEAK ACID/BASE SUBGROUP FOR IMDM MEDIUM                                       COMPONENT          mg/L                                                       ______________________________________                                        BIOTIN             0.0130                                                     CHOLINE CHLORIDE   4.0000                                                     FOLIC ACID         4.0000                                                     i-INOSITOL         7.2000                                                     NIACINAMIDE        4.0000                                                     PYRIDOXAL.HCL      4.0000                                                     RIBOFLAVIN         0.4000                                                     VITAMINE B.sub.12  0.0130                                                     D-GLUCOSE          4500.000                                                   HEPES              5958.0000                                                  PHENOL RED         15.0000                                                    SODIUM PYRUVATE    110.0000                                                   KCL                330.0000                                                   KNO.sub.3          0.0760                                                     NaSeO.sub.3 5H.sub.2 O                                                                           0.0173                                                     ______________________________________                                    

                  TABLE 18                                                        ______________________________________                                        GLUTAMINE CONTAINING SUBGROUP FOR IMDM MEDIUM                                 COMPONENT          mg/L                                                       ______________________________________                                        L-GLUTAMINE        584.00                                                     D-CA PANTOTHENATE  4.00                                                       CaCl.sub.2 (anhyd) 165.0                                                      MgSO.sub.4 (anhyd) 97.67                                                      NaCl               4505.00                                                    ______________________________________                                    

Modifications of the above-described modes for carrying out theinvention that are obvious to persons of skill in the media andfermentation art and/or related fields are intended to be within thescope of the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practice within the scope of the appended claims.

What is claimed is:
 1. A method of preparing a 1× to 10× mediumformulation, comprising(a) dividing the ingredients of said medium intotwo to six concentrated subgroups of compatible ingredients, wherein(i)one required compatible subgroup is an acid soluble subgroup having a pHof about 0 to about 1, and consisting essentially of at least one aminoacid or a derivative thereof and, optionally, at least one additionalingredient selected from the group consisting of thiamine, ascorbicacid, a ferric salt, a ferrous salt, guanine, adenine, glutathione, anda monobasic alkali phosphate, with the proviso that glutamine isexcluded from said acid soluble subgroup, (ii) a second requiredcompatible subgroup is a weak acid-base soluble subgroup having a pH inthe range of from about 4 to 9, and consisting essentially of at leastone ingredient selected from the group consisting of a sugar, anucleoside, a nucleotide, riboflavin, potassium chloride, sodiumchloride, a trace metal, a lipid, an acetate salt, a phosphate salt,HEPES, phenol red, a pyruvate salt, a buffer, a water soluble vitaminother than thiamine, ascorbic acid, and Ca-pantothenate; and wherein theremaining subgroups of compatible ingredients comprise:(iii) aglutamine-containing subgroup consisting essentially of at least oneingredient selected from the group consisting of D-Ca pantothenate,sodium chloride and glutamine stabilized with one or more divalentcations; (iv) an alcohol soluble subgroup consisting essentially of atleast one ingredient selected from the group consisting of cholesterol,a steroid, a fat soluble vitamin, a fatty acid, a lipid, TWEEN 80 and2-merpatoethanol; (v) an alkali soluble subgroup consisting essentiallyof at least one ingredient selected from the group consisting of purinesand pyrimidines that are stable and soluble at an alkaline pH; and (vi)a supplement-containing subgroup consisting essentially of at least oneingredient selected from the group consisting of an antibiotic, serum,yeastolate ultrafiltrate, typtose phosphate broth, insulin, transferrin,a growth factor, a hormone, albumin, whole egg ultrafiltrate, anattachment factor and sodium bicarbonate; and (b) admixing a sufficientamount said subgroups with a sufficient amount of a diluent to producesaid 1× to 10× cell medium formulation.
 2. The method of claim 1,wherein said subgroups are 10×-100×.
 3. The method of claim 1, whereinsaid amino acid or derivative thereof is selected from the groupconsisting of Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
 4. The method of claim 1,wherein said divalent cations are selected from the group consisting ofcalcium and magnesium.
 5. The method of claim 1, wherein the ratio ofglutamine to divalent cations is in the range of from 2:1 to 2.9:1. 6.The method of claim 1, wherein the ingredients of the medium are dividedinto three subgroups.
 7. The method of claim 1, wherein the ingredientsof the medium are divided into four subgroups.
 8. The method of claim 1,wherein the ingredients of the medium are divided into five subgroups.9. The method of claim 1, wherein the ingredients of the medium aredivided into six subgroups.
 10. The method of claim 1, wherein thesubgroups are greater than 10×.
 11. The method of claim 1, wherein thesubgroups are about 50×.
 12. The method of claim 1, wherein thesubgroups are about 100×.
 13. The method of claim 1, wherein saidadmixing is accomplished in a batch system.
 14. The method of claim 1,wherein said admixing is accomplished in a continuous system.
 15. Themethod of claim 1, wherein said diluent is water.
 16. The method ofclaim 1, wherein the concentrated subgroups are sterile.
 17. The methodof claim 1, wherein said medium formulation is 10×.
 18. The method ofclaim 1, wherein said medium formulation is 1×.
 19. A method ofpreparing a 1× to 10× medium formulation, comprising(a) dividing theingredients of said medium into two to six subgroups of compatibleingredients, wherein(i) one required subgroup is an acid solublesubgroup consisting essentially of at least one amino acid or aderivative thereof and, optionally, at least one additional ingredientselected from the group consisting of thiamine, ascorbic acid, a ferricsalt, a ferrous salt, guanine, adenine, glutathione, and a monobasicalkali phosphate, with the proviso that glutamine is excluded from saidacid soluble subgroup, (ii) a second required subgroup is a weakacid-base soluble subgroup consisting essentially of at least oneingredient selected from the group consisting of a sugar, a nucleoside,a nucleotide, riboflavin, potassium chloride, sodium chloride, a tracemetal, a lipid, an acetate salt, a phosphate salt, HEPES, phenol red, apyruvate salt, a buffer, a water soluble vitamin other than thiamine,ascorbic acid, and Ca-pantothenate; and wherein the remaining subgroupsof compatible ingredients comprise:(iii) a glutamine-containing subgroupconsisting essentially of at least one ingredient selected from thegroup consisting of D-Ca pantothenate, sodium chloride and glutaminestabilized with one or more divalent cations; (iv) an alcohol solublesubgroup consisting essentially of at least one ingredient selected fromthe group consisting of cholesterol, a steroid, a fat soluble vitamin, afatty acid, a lipid, TWEEN 80 and 2-merpatoethanol, (v) an alkalisoluble subgroup consisting essentially of at least one ingredientselected from the group consisting of purines and pyrimidines that arestable and soluble at an alkaline pH, and (vi) a supplement-containingsubgroup consisting essentially of at least one ingredient selected fromthe group consisting of an antibiotic, serum, yeastolate ultrafiltrate,typtose phosphate broth, insulin, transferrin, a growth factor, ahormone, albumin, whole egg ultrafiltrate, an attachment factor andsodium bicarbonate; and (b) dissolving each of said subgroups in acarrier to give 10× to 100× concentrated solutions of said subgroups inthe respective carrier; (c) adjusting, if necessary, the pH's of thesubgroups so that:(i) the pH of the acid soluble subgroup is about 0 toabout 1; (ii) the pH of the weak acid-base soluble subgroup is in therange of from about 4 to 9; (iii) the pH of the alkali-soluble subgroupis at about 9.0; (d) after adjusting the pH's if necessary, admixing asufficient amount of said subgroups with a sufficient amount of adiluent to give said 1× to 10× medium.
 20. The method of claim 19,wherein said subgroups are 10×-100×.
 21. The method of claim 19, whereinsaid amino acid or derivative thereof is selected from the groupconsisting of Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
 22. The method of claim 19,wherein said divalent cations are selected from the group consisting ofcalcium and magnesium.
 23. The method of claim 19, wherein the ratio ofglutamine to divalent cations is in the range of from 2:1 to 2.9:1. 24.The method of claim 19, wherein the pH of the glutamine containingsubgroup is in the range of 4.0 to 6.0.
 25. The method of claim 19,wherein the ingredients of the medium are divided into three subgroups.26. The method of claim 19, wherein the ingredients of the medium aredivided into four subgroups.
 27. The method of claim 19, wherein theingredients of the medium are divided into five subgroups.
 28. Themethod of claim 19, wherein the ingredients of the medium are dividedinto six subgroups.
 29. The method of claim 19, wherein the subgroupsare greater than 10×.
 30. The method of claim 19, wherein the subgroupsare about 50×.
 31. The method of claim 19, wherein the subgroups areabout 100×.
 32. The method of claim 19, wherein the medium is a 10×medium formulation.
 33. The method of claim 19, wherein the medium is a1× medium formulation.
 34. The method of claim 19, wherein said admixingis accomplished in a batch system.
 35. The method of claim 19, whereinsaid admixing is accomplished in a continuous system.
 36. The method ofclaim 19, wherein said carder and said diluent are water.
 37. The methodof claim 18, wherein said subgroups are sterile.
 38. A kit for preparinga 1× to 10× medium formulation comprising two to six containers, whereineach of said containers comprises a subgroup of concentrated compatibleingredients for preparing said formulation, wherein(i) one firstrequired subgroup is an acid soluble subgroup having a pH of about 0 toabout 1, and consisting essentially of at least one amino acid or aderivative thereof and, optionally, at least one additional ingredientselected from the group consisting of thiamine, ascorbic acid, a ferricsalt, a ferrous salt, guanine, adenine, glutathione, and a monobasicalkali phosphate, with the proviso that glutamine is excluded from saidacid soluble subgroup; (ii) a second required subgroup is a weakacid-base soluble subgroup having a pH in the range of from about 4 to9, and consisting essentially of at least one ingredient selected fromthe group consisting of a sugar, a nucleoside, a nucleotide, riboflavin,potassium chloride, sodium chloride, a trace metal, a lipid, an acetatesalt, a phosphate salt, HEPES, phenol red, a pyruvate salt, a buffer, awater soluble vitamin other than thiamine, ascorbic acid, andCa-pantothenate; and wherein the remaining subgroups comprise: (iii) aglutamine-containing subgroup consisting essentially of at least oneingredient selected from the group consisting of consisting essentiallyof D-Ca pantothenate, sodium chloride and glutamine stabilized with oneor more divalent cations; (iv) an alcohol soluble subgroup consistingessentially of at least one ingredient selected from the groupconsisting of cholesterol, a steroid, a fat soluble vitamin, a fattyacid, a lipid, TWEEN 80 and 2-merpatoethanol, (v) an alkali solublesubgroup consisting essentially of at least one ingredient selected fromthe group consisting of purines and pyrimidines that are stable andsoluble at an alkaline pH of about 9.0, and (vi) a supplement-containingsubgroup consisting essentially of at least one ingredient selected fromthe group consisting of an antibiotic, serum, yeastolate ultrafiltrate,typtose phosphate broth, insulin, transferrin, a growth factor, ahormone, albumin, whole egg ultrafiltrate, an attachment factor andsodium bicarbonate.
 39. The kit of claim 38, wherein said amino acid orderivative thereof is selected from the group consisting of Ala, Arg,Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, and Val.
 40. The kit of claim 38, wherein said divalentcations are selected from the group consisting of calcium and magnesium.41. The kit of claim 38, wherein the ratio of glutamine to divalentcations is in the range of from 2:1 to 2.9:1.
 42. The kit of claim 38,wherein said glutamine-containing subgroup has a pH in the range of 4.0to 6.0.
 43. The kit of claim 38, wherein said kit comprises threecontainers.
 44. The kit of claim 38, wherein said kit comprises fourcontainers.
 45. The kit of claim 38, wherein said kit comprises fivecontainers.
 46. The kit of claim 38, wherein said kit comprises sixcontainers.
 47. The kit of claim 38, wherein said subgroups are greaterthan 10×.
 48. The kit of claim 38, wherein subgroups are about 50×. 49.The kit of claim 38, wherein said subgroups are sterile.
 50. A method ofstabilizing glutamine in solution, comprising admixing a solution ofglutamine with one or more divalent cations.
 51. The method of claim 50,wherein said divalent cations are selected from the group consisting ofcalcium and magnesium.
 52. The method of claim 50, wherein said divalentcations are supplied as part of a salt.
 53. The method of claim 52,wherein said salt is MgCl₂, CaCl₂, Ca(NO₃)₂ or MgSO₄.
 54. The method ofclaim 50, wherein the ratio of glutamine to divalent cations is in therange 2:1 to 2.9:1.